The role of mucosal innate immunity in allergic rhinitis Ji-Hwan Ryu
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1 The role of mucosal innate immunity in allergic rhinitis Ji-Hwan Ryu Research Center for Human Natural Defense system College of Medicine Yonsei University
2 Mucosal epithelium in innate immunity
3 Mucosal epithelium in innate immunity
4 Nasal epithelium Human nasal epithelium
5 Innate immunity components of airway epithelium
6 Reactive oxygen species (ROS) act as second messenger e e Catalase/peroxidase O 2 H 2 O 2. H 2 O SOD (mild) (mild) e OH e O 2. - (toxic) Cytokines Growth factors GPCR ligands UV and X-ray Pathophysiology Mitochondria NADPH oxidases High Low Quiescence Proliferation _. O H 2 O 2 2 Apoptosis Necrosis
7 Transmembrane topology of NADPH oixdase isozymes Lambeth JD, Nature Rev. Immunol. 4, 181, 24
8 Function of DUOX-mediated ROS in airway epithelial cells -DUOX 1 regulates MUC5AC mucin expression in human airway epithelial cells (Shao et al., PNAS, 25) - DUOX1 regulates production of inflammatory mediators such as IL-8 in response to environmental or bacterial stimuli (Koff et al., Am J Physiol Lung Cell Mol Physiol, 28), (Boots et al., JBC, 29) - DUOX2 is the major contributor to H 2 O 2 synthesis induced by bacterial stimuli (Gattas et al., Free Radic Biol Med, 29)
9 Role of ROS in allergic airway inflammation - ROS generated by Pollen NADPH oxidase augments allergic airway inflammation (Boldogh et al., J Clin Invest, 25) -Tyrosine phosphatase SHP-1 regulates development of allergic airway inflammation by controlling intracellular ROS level (Cho et al., Am J Respir Cell Mol Biol, 28) -Level of H 2 O 2 is increased in exhaled breath from patients with inflammatory airway diseases (Baldwin et al., Lancet, 1986), (Kietzmann et al., Intensive Care Med, 1993), (Fischer, Antioxidants and Redox Signaling, 29)
10 Schematic of innate immune mechanisms activated by allergens TLR4 (M Wills-Karp, A Nathan, CL Karp, Mucosal Immunology, 21)
11 The role of Innate immunity in allergic inflammation House dust mite Innate immunity ROS? Airway epithelial cell membrane Allergic airway inflammation
12 Airway liquid interface (ALI) culture for human nasal epithelial cell Air Medium
13 HDM stimulates ROS production in Normal Human Nasal Epithelial (NHNE) cells Relative DCF fluorescence (fold over control) HDM (5 µg/ml) (min) (min) HDM (5 µg/ml)
14 Main components of house dust mite extract House dust mite (Dermatophagoides pteronyssinus) Group Biochemical identity Molecular weight (kda) Allergen 1 Cysteine protease 25 Der p1 2 Niemann-Pick C2 homologue 14 Der p2 3 Trypsin 25 Der p3 4 Alpha-amylase 56 Der p4 5 Unknown 15 Der p5 6 Chymotrypsin 25 Der p6 7 Unknown 24 Der p7 8 Glutathione S-transferase 26 Der p8 9 Collagenolytic serine protease 29 Der p9 1 Tropomyosin 35 Der p1 11 Paramyosin 1 Derp11
15 HDM-induced ROS production is not dependent on protease Relative DCF fluorescence (fold over control) Cont HDM HDM+PI Papain Trypsin Cont HDM HDM+PI Trypsin Papain
16 Relative DCF fluorescence (fold over control) HDM-induced ROS production is not dependent on LPS
17 Relative DCF fluorescence (fold over control) b-glucans in HDM induce ROS production
18 Relative DCF fluorescence (fold over control) b-glucan structure such as curdlan induces ROS production Curdlan (1 µg/ml) min min Curdlan (1 µg/ml)
19 Relative DCF fluorescence (fold over control) b-glucan structure such as zymosan induces ROS production (min) Zymosan (1 µg/ml)
20 The role of Innate immunity in allergic inflammation House dust mite b-glucan Innate immunity ROS Airway epithelial cell membrane Mediator of Innate immunity? Allergic airway inflammation
21 Interaction between epithelial cells and Dendritic cells (DCs) in allergic inflammation - β-glucan in HDM induces CCL2 production in human bronchial epithelial cell line (Nathan et al., J Allergy Clin Immunol, 29) - Production of CCL2 induces recruitment of myeloid DCs and memory CCR6+CD4+ cells into the airways, which lead to allergic asthma (Lukacs et al., J Exp Med, 21) - CCL2 expression level is up-regulated in nasal mucosa from allergic rhinitis patients (Wu et al., Am j of otolaryngology, 29) Clare M LIoyd. Nature Medicine (29)
22 HDM-induced ROS production is responsible for CCL2 secretion CCL2 (pg/ml) CCL2 (pg/ml) (hr) HDM (5µg/ml) Cont HDM HDM+ NAC
23 b-glucan-induced ROS production is responsible for CCL2 secretion CCL2 (pg/ml) Curdlan (µg/ml) Zymosan (µg/ml)
24 Signaling pathway involved in b-glucan-induced ROS production and CCL2 secretion House dust mite b-glucan Airway epithelial cell membrane? Recognition receptor? NADPH oxidase? ROS CCL2 secretion Allergic airway inflammation
25 Relative DCF fluorescence (fold over control) Curdlan-induced ROS production is mediated by TLR2 Cont sirna TLR1 sirna TLR2 sirna TLR4 sirna TLR6 sirna Dectin1 sirna urdlan Cont Cont Cont sirna TLR1 sirna TLR2 sirna TLR4 sirna TLR6 sirna Dectin1 sirna Curdlan
26 CCL2 (pg/ml) Curdlan-induced CCL2 secretion is mediated by TLR Cont Cont Cont sirna TLR1 sirna TLR2 sirna TLR4 sirna TLR6 sirna Dectin1 sirna Curdlan
27 Relative DCF fluorescence (fold over control) Relative DCF fluorescence (fold over control) DUOX2 is responsible for curdlan-induced ROS production Curdlan Curdlan
28 CCL2 (pg/ml) DUOX2 is responsible for curdlan-induced CCL2 secretion Cont Cont Cont sirna NOX1 sirna NOX2 sirna NOX4 DUOX1 sirna sirna DUOX2 sirna Curdlan
29 TLR2/DUOX2 regulate the ROS production and consequent CCL2 secretion in primary human nasal epithelial cells Relative DCF fluorescence (fold over control) CCL2 (pg/ml) Cont shrna TLR2 shrna DUOX2 shrna Cont shrna TLR2 shrna DUOX2 shrna PBS Curdlan PBS Curdlan
30 DCs recruitment in mouse nasal epithelium House dust mite b-glucan Airway epithelial cell membrane TLR2 ROS DUOX2 CCL2 secretion In vivo mouse model Dendritic cell recruitment? Allergic airway inflammation
31 b-glucans within HDM are responsible for CCL2 secretion, and nasal mucosa DCs (NMDCs) recruitment CCL2 (pg/ml) 25 PBS HDM HDM+ polymyxin B μm HDM+ b-glucanase HDM+PI Immunohistochemistry with anti-cd11c antibody
32 b-glucans within HDM are responsible for NMDCs recruitment Percentage of MHCII + CD11c + DCs (%)
33 TLR2 is required for curdlan or HDM-induced CCL2 secretion and NMDCs recruitment CCL2 (pg/ml) Percentage of MHCII + CD11c + DCs (%) TLR2 +/+ TLR2 -/- TLR2 +/+ TLR2 -/ PBS Curdlan HDM PBS Curdlan HDM
34 ROS are mediated for curdlan or HDM-induced CCL2 secretion and NDDCs recruitment CCL2 (pg/ml) Percentage of MHCII + CD11c + DCs (%) WT WT + NAC WT WT + NAC PBS Curdlan HDM PBS Curdlan HDM
35 CCL2 (pg/ml) DUOX2 is required for curdlan or HDM-induced CCL2 secretion and NDDCs recruitment Percentage of MHCII + CD11c + DCs (%) DUOX2 +/+ DUOX2 +/thyd DUOX2 thyd/thyd DUOX2 +/+ DUOX2 +/thyd DUOX2 thyd/thyd PBS Curdlan HDM PBS Curdlan HDM
36 b-glucan in HDM induces innate immune response through TLR2/DUOX2-mediated ROS production House dust mite b-glucan Airway epithelial cell membrane TLR2 ROS DUOX2 CCL2 secretion DCs recruitment Allergic rhinitis model Allergic airway inflammation? (Allergic rhinitis?)
37 Allergic diseases Allergic disease is the 5th leading chronic disease among all ages 3rd common chronic disease among children under 18 years old; up to one child in three is affected Trends indicate that by 215, half of all westerns may be suffering from an allergy
38 Allergic rhinitis Definition Rhinitis is an inflammation of the mucous membranes of the nose. Allergic rhinitis involves reactions in the nasal mucosa from repeated allergen exposures that causes hypersensitivity Systemic symptoms of allergic rhinitis - Runny nose - Nasal obstruction - sneezing - Fatigue - Difficulty concentrating - Decreased appetite
39 Allergic rhinitis and coexistent airway diseases Otitis Media Upper Respiratory infection Allergic rhinitis Nasal Polyposis Asthma Sinusitis
40 Research needs on allergic rhinitis and asthma To identify the causes of the increasing prevalence, which might lead to prevention - risk factors, epidemiology- To define the exact mechanisms causing allergic symptoms, which could lead to better treatments - disease mechanisms, management-
41 Protocol diagrams for the induction of allergic rhinitis mouse model using HDM extract HDM extract (1 µg) + aluminium hydroxide (2 mg) PBS control or HDM extract (5 µg) Day Intraperitoneal (i.p.) sensitization Intranasal (i.n.) challenges Sacrifice
42 Verification of allergic rhinitis mouse model Isolation of nasal mucosa Immunohistochemistry Flow cytometry analysis HDM sensitization/ PBS challenges HDM sensitization/ HDM challenges HDM sensitization/ PBS challenges Isolation of cervical lymph node Cell culture Restimulation With PBS 4 days incubation ELISA quantification of Th2 cell cytokines HDM sensitization/ HDM challenges Restimulation With HDM
43 Allergic inflammation in the lung is not induced in our allergic rhinitis model Percentage of CCR3 + CD3 - eosinophils (%) (pg/ml) (pg/ml) (pg/ml) (pg/ml) IL-4 5 IL HDM/PBS HDM/HDM HDM/PBS HDM/HDM HDM/PBS HDM/HDM IL IFN-γ HDM/PBS HDM/HDM HDM/PBS HDM/HDM
44 b-glucan in HDM promote HDM-induced allergic rhinitis Counts/15 minutes Percentage of CCR3 + CD3 - eosinophils (%)
45 b-glucan in HDM promote HDM-induced allergic rhinitis IL- 13 (pg/ml) IL- 4 (pg/ml) IL- 5 (pg/ml) IFN- γ (pg/ml)
46 Counts/15 minutes Percentage of CCR3 + CD3 - eosinophils (%) TLR2 and DUOX2 required for HDM-induced allergic rhinitis TLR2 +/+ TLR2 -/- DUOX2 +/+ DUOX2 thyd/thyd HDM/PBS HDM/HDM TLR2 +/+ TLR2 -/- DUOX2 +/+ DUOX2 thyd/thyd HDM/PBS HDM/HDM
47 IL- 13 (pg/ml) IFN- γ (pg/ml) IL- 4 (pg/ml) IL- 5 (pg/ml) TLR2 and DUOX2 required for HDM-induced allergic rhinitis TLR2 +/+ TLR2 -/- DUOX2 +/+ DUOX2 thyd/thyd TLR2 +/+ TLR2 -/- DUOX2 +/+ DUOX2 thyd/thyd HDM/PBS HDM/HDM HDM/PBS HDM/HDM TLR2 +/+ TLR2 -/- DUOX2 +/+ DUOX2 thyd/thyd TLR2 +/+ TLR2 -/- DUOX2 +/+ DUOX2 thyd/thyd HDM/PBS HDM/HDM HDM/PBS HDM/HDM
48 Summary
49 Acknowledgments Research Center for Human Natural Defense system, Yonsei University College of Medicine Dr. Joo-Heon Yoon Dr. Chang-Hoon Kim Dr. Jung Hee Joo Jung-Yeon Yoo Min-Ji Kim Kwang Chul Ahn Sang-Gyu Hwang Department of Life Sciences Ewha Womans University Dr. Yun Soo Bae Dr, Jaesang Kim Dr, Won-Jae Lee Jee Hyun Lee Mucosal Immunology Section, International Vaccine Institute Dr. Mi-Na Kweon Doo-Hee Shim Department of Oral Biology, Yonsei University College of Dentistry Dr. Dong Min Shin Arthropods of Medical Importance Resource Bank, Yonsei Universigy College of Medicine Dr. Tai-Soon Yong Dr. Kyoung Yong Jeong
50 Thank you for your attention!
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